CN101134976A - Method for producing validamycin by circulating fermentation - Google Patents
Method for producing validamycin by circulating fermentation Download PDFInfo
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- CN101134976A CN101134976A CNA2006101126712A CN200610112671A CN101134976A CN 101134976 A CN101134976 A CN 101134976A CN A2006101126712 A CNA2006101126712 A CN A2006101126712A CN 200610112671 A CN200610112671 A CN 200610112671A CN 101134976 A CN101134976 A CN 101134976A
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Abstract
The present invention is circulating fermentation process for producing jinggangmycin. The circulating fermentation process has the fermented residue and effluent reused completely in the culture medium of the next time. The present invention has the advantages of no discharged waste slag and drained waste water.
Description
Technical field
The present invention relates to a kind of production method of biological pesticide, specifically a kind of novel method by the fermentative production jingganmycin.
Background technology
Jingganmycin is the secondary metabolite that a streptomycete that China scientist 1972 finds from the soil of area, Jinggang Mountain is produced, and is the agricultural antibiotic of China's independent development.Jingganmycin has been made major contribution to China's rice high yield stable yields over surplus in the of 30 year, has become the widely known desirable biological pesticide of China peasant.
Jingganmycin is produced by the mutation of streptomyces hygroscopicus well ridge, and close with the validamycin validamycin A of Japan, the two all is made up of 8 main ingredients such as A, B, C, D, E, F, G, H, and its most important component is a jinggangmycin A.Jinggangmycin A belongs to efficient, low toxicity sterilant, and the lasting period is long, and resistance of rainwater washing against is safe in utilization, and noresidue is to the people, animal low toxicity, to fish, honeybee and natural enemies security, free from environmental pollution.
The jingganmycin chemical name is: N-((1S)-(1,4,6/5)-3-methylol-4,5,6-trihydroxy--2-is hexenyl also)-(O-β-D-glucopyranosyl-(1 → 3)-(1S)-(1,2,4/3,5)-2,3,4-trihydroxy--5-methylol hexahydroaniline; English name is: jinggangmycinA; Validamycin A; Validacin; Valimon.Structure or molecular formula are:
Relative molecular weight: 515.51, there is not certain fusing point, 95～100 ℃ are softening, about 135 ℃ of decomposition.
Jingganmycin is the low toxicity sterilant.It is the very strong agricultural antibiotic of systemic action.Generally being processed as aqua, water solube powder or pulvis uses.Mainly prevent and treat rice sheath blight disease, rice green smut, wheat sclerotium disease, the leaf blight of corn, vegetables damping-off, cotton, beans damping-off, from beautiful disease, genseng damping-off etc.
Jingganmycin is normally done the substratum fermentation with agricultural and sideline tight product and is formed.Fermented residue (being called for short the well slag) after filtering because acid big (about PH3); Moisture height (more than 70%); Viscosity is big; Be not easy to handle, cause environmental pollution.The whole nation produces that jingganmycin is pure 5000 tons per year, and producing the well slag has more than 60,000 ton.Although some producer is used as feed with the oven dry of well slag or pulverizes and directly mix in the pulvis of well ridge, because its moisture content height, viscosity is big, stinks to high heaven, and dealing with needs a large amount of energy, and many producers are unwilling to handle.Some well ridge manufacturing enterprises feed fish directly for local peasant in waste residue and feed pigs, may bring negative impact to food safety like this.That have even direct the discharging caused environmental pollution.In the production process of well ridge, its last handling process can produce a large amount of waste water in addition, and the amount of these waste water is bigger than well slag, and the pollution of environment is surpassed the well slag, if do not handle, can cause serious environmental to pollute.
Summary of the invention
At bacterium slag, the waste water environmental pollution problem of jingganmycin fermentation generation, the invention provides the novel method of producing validamycin by circulating fermentation.
Production method of the present invention be with on take turns zymophyte slag and waste water as one of substratum of fermentation culture next time, repeat repeatedly to utilize.Its concrete scheme is as follows:
1. seed preparation
1.1 bacterial classification: the streptomyces hygroscopicus jingganmycin produces bacterium (Strptomyces hygroscopicusvar.Jingganggensis yen), is preferably the NFY-1022 bacterial strain, and its preserving number is CCTCCM206074.
1.2 substratum
1.1.1. slant medium (female inclined-plane and eggplant bottle inclined-plane)
Glucose 1.0% asparagine 0.05% KH
2PO
40.05%
Agar 1.5-1.6%pH value nature
1.1.2 seed culture medium
Rice meal 4.0% peanut meal powder 1.0% yeast powder 0.5%
Bean cake powder 1.0% peptone powder 0.5% KH
2PO
40.025%
CaCO
30.3% NaCl, 0.2% pH value nature
1.3 seed preparation process:
Under aseptic condition, the streptomyces hygroscopicus jingganmycin is produced the female inclined-plane of bacterium sandy soil spore inoculating, cultivated 5～7 days for 26～30 ℃, wait to grow switching eggplant bottle inclined-plane behind the abundant grey spore, cultivated 5～6 days for 26～30 ℃, jar spore suspension is advanced in preparation after waiting to grow abundant grey spore, and the inoculation seeding tank, carries out seed culture.
Seed tank culture condition: temperature: 39～40 ℃, tank pressure: 0.05mpa, air flow quantity: 1: 1.2～1: 1.5v/v/min, stir: 150～250 rev/mins, incubation time: 22～24 hours.
1.4 corresponding seed quality criteria
1.4.1 the seed liquid appearance is filbert, evenly, no granule foreign, the microscopy mycelia is netted gathers, and does not have assorted bacterium, mycelia even dyeing, pH5.5-6.8.
2. jingganmycin fermentation culture:
2.1 fermention medium (W/W)
Starchy material (as Starch rice, W-Gum, potato starch etc.) 5～20%, wet bacteria slag (water content 50～85%) 1～30%, soybean cake powder 0.1～5%, groundnut meal 0.1～5%, yeast powder 0.5～5%, lime carbonate 0.5～5%, on take turns fermentation waste water, deficiency is supplied with tap water, regulates pH6.5～8.0.
2.2 fermentor cultivation condition and management (technology controlling and process)
2.2.1 temperature: 39-42 ℃, be preferably 40 ℃, patrolled and examined once in per 15 minutes, note keeping temperature-stable.
2.2.2. it is serious that tank pressure 0.01mpa～0.06mpa bubbled as the fermentation later stage, can suitably improve tank pressure to 0.06mpa.
2.2.3 air flow quantity: 0～8 hour, 1: 0.5～1: 1.0v/v/min; Then to putting jar 1: 1.2v/v/min
2.2.4 culture transferring amount (by kind of daughter bacteria liquid and fermentor cultivation liquid volume ratio): 5%～10%
2.2.5 stirring velocity: can not open stirring in 0～6 hour; 100～300rpm after 6 hours.
2.2.6, notice that foam increases according to the fermentation situation, add bubble enemy froth breaking in good time, notice that the top jar is not escaped liquid.
2.2.7 record: per hour once jar is warm for record, air, flow and tank pressure.
2.2.8 sampling: 24 hours mid-early stages of fermenting process every sampling in 8 hours once, carry out sterility test and serial chemical analysis, survey pH, total reducing sugar, amino nitrogen, cell concentration, filtration velocity, microscopy mycelial growth situation has or not microbiological contamination, begin to survey fermentation unit after 24 hours, sampling every two hours once after 24 hours.
2.2.9 fermentation time 32～50 hours.
2.3 normally put a jar standard
2.3.1 fermentation unit A component〉18000 μ g/ml.
2.3.2 fermented liquid residual sugar＜2.0%; Reducing sugar＜1.5%.
2.3.3 fermented liquid filtering velocity〉10ml/min.
2.3.4 the fracture of bacterium mirror mycelia, cavity are in flakes big, pH begins to rise to about 7.0.
3. post-processed
With fermented liquid by the plate-and-frame filter press press filtration, filtrate, filtrate again through concentrate drying and finished product.Water of condensation when the waste water of the bacterium slag that filter press obtains, the waste water that cleans filter cloth, cleaning fermentor tank and filtrate concentrate drying together enters the material-compound tank making beating with fresh feed, enters fermentor tank sterilization back again and uses (referring to Fig. 2).
Fermentation process of the present invention has been eliminated the discharging of bacterium slag and waste water with fermentation residue and waste water circulation recycling, has reduced production cost, meets the trend of current creation of resources conservation-minded society.
Description of drawings
Fig. 1 is a seed preparation technology schema;
Fig. 2 is a jingganmycin circulating fermentation production scheme.
Specific embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
The preparation and the fermentation culture of embodiment 1 seed
1. seed preparation
1.1 bacterial classification: well ridge streptomycete NFY-1022 bacterial strain
1.2 substratum
1.1.1。Slant medium (female inclined-plane and eggplant bottle inclined-plane, each percentage composition in the substratum is weight percentage, down together)
Glucose 1.0% asparagine 0.05% KH
2PO
40.05%
Agar 1.6% pH value nature
1.1.2 seed culture medium
Rice meal 4.0% peanut meal powder 1.0% yeast powder 0.5%
Bean cake powder 1.0% peptone powder 0.5% KH
2PO
40.025%
CaCO
30.3% NaCl, 0.2% pH value nature
1.3 seed preparation technology flow process is seen accompanying drawing 1:
1.4 the preparation of slant medium
1.4.1 weighing and calculating: require in proportion earlier to calculate, take by weighing respectively then. preparation inclined-plane water should be distilled water or synthetic water, and agar is with agar powder or good agar strip.
1.4.2 molten joining and loading amount: earlier the glucose water is dissolved, mix mutually with other medicine, the test tube slant substratum is with 180 * 18mm test tube.Every about 6-7ml substratum of dress, the bottled amount of eggplant is 70ml.
1.4.3 sterilization: pressure is 0.08mpa, 118 ℃ of temperature treat that it is cooled to 70-80 ℃ and puts the inclined-plane after the time 30min sterilization.
1.5 slant culture and the preparation of advancing jar spore suspension
1.5.1 test tube slant seed preparation: under aseptic condition, with a little sandy soil spore of aseptic inoculation ring picking, uniformly dispersing is accused on the inclined-plane, to be coated with notice even as far as possible, cultivated 6 days for 28 ℃, wait to grow switching eggplant bottle inclined-plane behind the abundant grey spore, place in 4 ℃ of refrigerators, can preserve one month.
1.5.2 eggplant bottle inclined-plane seed preparation: get above-mentioned test tube slant spore, scrape with transfering loop and to get a ring spore, be uniformly coated on the eggplant bottle inclined-plane, cultivated 5-6 days for 28 ℃, jar spore suspension is advanced in preparation after waiting to grow abundant grey spore, and this eggplant bottle can be placed in the refrigerator and preserve about 10 days.1.5.3 advance the preparation of jar spore suspension: the eggplant phialosporae grow good after, every bottle adds the 100ml sterilized water, scraping spore is spore suspension, many phialosporaes suspension is transferred to (inoculum size is 1M in the inoculation bottle
3Seed liquor is with an eggplant bottle) use as the inoculation seeding tank.
1.6 the preparation of seed tank culture base
1.6.1 calculate the requirement and the weighing of each raw material by the requirement of culture medium prescription, some granular raw materials pulverized earlier, or dissolve with less water.
1.6.2 in fermentor tank (or material-compound tank), add 1/3 earlier with water.
1.6.3 opening stirring (or ventilation) material that proportioning is good drops in the jar.
1.6.4 calculated the amount of water of condensation in the sterilization process, supplied water and regulate the pH value.
1.6.5 open the preheating of chuck (or coiled pipe) steam inlet valve.
1.6.6 cleaning dog-house and tank top.Stir, inoculation mouthful is cleared up standbyly, closes dog-house.
1.7 sterilization
1.7.1 drive the jacket steam valve, the interlayer preheating.
The air valve 1.7.2 begin to rehearse is suitably opened the inoculation valve, so that exhaust is unimpeded.
Close the interlayer steam valve more than 90 ℃ 1.7.3 a jar temperature reaches, stop stirring.
1.7.4 the compensation flap on the air filter of pass is opened air filter base bleed valve, makes strainer pressure drop to zero.
1.7.5 drive the steam valve on the air filter, treat that condensation changes the back and closes strainer below vapor cock, make strainer pressure maintain 0.5mpa.
1.7.6 drive the steaming soil valve on the sampling line, drive sampler second valve and bleed off water of condensation, treat emptying after, turn down the little exhaust of thief hole second valve, open thief hole first valve.
1.7.7 drive the steam valve on the material pipe, drive road wash water valve under the materail tube, treat to close valve after the cold water emptying, open material and advance pot valve.
1.7.8 open towards the visor steam valve.
1.7.9 open the steam valve of culture transferring pipeline, in a word, should guarantee with jar in the various pipeline valve steam that are connected unimpeded, dead angle or short circuit must not be arranged, treat that tank pressure is raised to 0.1～0.12mpa, temperature picks up counting when being raised to 118 ℃, and 121 ℃ again～123 ℃ of holding temperatures, keep 30min.Notice that temperature-rise period is unsuitable long, should be rapidly heated.
1.7.10 the steam of closing on the air filter advances pot valve, drives pressure lock when device pressure to be filtered is lower than total filter pressure, drives big equilibrium valve, makes the air blow drying strainer.
1.7.11 close sampling first valve.
1.7.12 close material first valve, close the steam valve of material top, close towards the visor valve inoculation mouth etc.
1.7.13 when tank pressure drops to 0.05mpa, close the strainer equilibrium valve, open air and go into pot valve, regulate intake valve and exhaust, keep tank pressure 0.05mpa.
1.7.14 open interlayer water supply valve (coiled pipe) cooling.
1.7.15 open mixing speed 190rpm.
1.7.16 when treating jar temperature drop to 50 ℃, close the interlayer water supply valve, inoculation when treating further to reduce to 40 ℃.
1.8 inoculation
1.8.1 close the doors and windows.
1.8.2 stop stirring.
1.8.3 the operator must wear mouth mask, cap, and gloves reduce air flowing as far as possible.
1.8.4 with cotton ball soaked in alcohol be placed on the inoculation mouthful around.
Advance pot valve 1.8.5 close air.
1.8.6 light the alcohol swab circle.
1.8.7 the pass vent valve makes tank pressure reduce to zero, closes vent valve.
The inoculation cap 1.8.8 slowly outward winding.
1.8.9 ready spore suspension is poured in the jar.
1.8.10 will inoculate on the cap, tighten rapidly, extinguish the alcohol swab circle.
Go into pot valve 1.8.11 open air, regulate vent valve, make it flow 1: 1.0～1: between the 1.2v/v/min, tank pressure is controlled at 0.05mpa.
1.8.12 open stirring, carry out seed and accompany foster.
1.9 seed tank culture condition
1.9.1 temperature: 39～40 ℃, tank pressure: 0.05mpa, air flow quantity: 1: 1.2～1: 1.5v/V/min, stir: 200 rev/mins, incubation time: 24 hours.
1.10 seeding tank management
1.10.1 jar temperature when higher or on the low side, manually add cold water formula hot water by interlayer and regulates, and is all when needing the regulating tank temperature, must not leave, and treats can leave behind the valve-off.
1.10.2 checked once, and should check the temperature of instrument temperature and glass-stem thermometer during inspection simultaneously, in per 15 minutes in order to avoid instrument malfunction.
1.10.3 when jar temperature abnormality raises or reduce, in time check hydraulic pressure and relevant devices, especially jar is gone up each steam valve, has leakage in time to take measures if find.
1.10.4 because the variation of total air consumption and pressure can cause flow rate fluctuation, should in time adjust, generally should control branch strainer air pressure at 0.13～0.15mpa.
1.10.5 per hour write down temperature, flow, air pressure, tank pressure and abnormal conditions and stress measure.
1.11 corresponding seed quality criteria
1.11.1 the seed liquid appearance is filbert, evenly, no granule foreign, the microscopy mycelia is netted gathers, and does not have assorted bacterium, mycelia even dyeing, pH5.5～6.8.
2. jingganmycin circulating fermentation production method:
2.1 fermention medium (W/W)
W-Gum 10%, wet bacteria slag (water content 70%) 20%, soybean cake powder 3%, groundnut meal 3%, lime carbonate 3%, on take turns fermentation waste water, not enough supply with tap water, sheet alkali is regulated pH7.0.
2.2 zymotechnique is seen accompanying drawing 2:
2.3 the preparation of fermentation tank culture medium
2.3.1 calculate the requirement of each raw material and weigh up in advance by the requirement of substratum, will go up wheel fermentation wet bacteria slag and waste water at the dosing chamber pulping.And add other raw material, squeeze into fermentor tank.
2.3.2 calculated the amount of water of condensation in the sterilization process, supplied water and regulate the pH value.
2.3.3 drive interlayer (or coiled pipe steam) inlet valve.
Should be about 7.6 2.3.4 check the pH value,, should be adjusted to this scope as not in this scope.
2.3.5 the cleaning dog-house, tank top and agitator are closed dog-house to clean.
2.3.6 when substratum is heated to 90 ℃, close (interlayer) coiled pipe or steam, stop to stir.
2.4 fermentor tank sterilization
Real jar of sterilization with reference to the seeding tank sterilization, after the fermentor tank sterilization, cools to 40 ℃ of culture transferrings.Notice that the temperature rise period (90～118 ℃) should be rapidly heated in the sterilization process, the time is unsuitable long, keeps material nutrition loss within reason, and sterilization back total reducing sugar should be controlled at more than 8.5%.
2.5 culture transferring
2.5.1 fermentor tank and seeding tank stopple coupon sterilization sampling after 30 minutes.Carry out biochemical analysis and sterility test.
2.5.2 drive culture transferring pipeline steam valve and inoculation valve steam valve.
2.5.3 drive fermentor tank culture transferring pipeline top valve, open vent valve.
2.5.4 drive seeding tank transferred species pipeline top valve, open vent valve.
2.5.5 guarantee sterilization 1 hour.
2.5.6 after the sterilization of culture transferring pipeline finishes, close the vapor cock on all fermentor tanks and the seeding tank culture transferring pipeline.
2.5.7 close the steam valve on the pipeline.
2.5.8 driving fermentor tank stirs.
Advance pot valve 2.5.9 open fermentor tank culture transferring pipeline.
2.5.10 close the seeding tank vent valve, to improve the seeding tank tank pressure.
2.5.11 open the fermentor tank vent valve, make tank pressure remain on 0.03mpa
2.5.12 open seeding tank culture transferring pipeline top valve.
Go into pot valve 2.5.13 regulate the fermentor tank air, utilize pressure reduction that seed is pressed into fermentor tank.
2.5.14 culture transferring finishes, and closes fermentor tank culture transferring pipeline and advances tank valve.
Go into pot valve 2.5.15 close the seeding tank air, make tank pressure fall zero.
2.5.16 regulate fermentation tank pressure and air flow, make it to be controlled at respectively 0.02～0.06mpa and 1: 1.0～1: 1.5v/v/min.
2.5.17 drive the steam valve of culture transferring pipeline top.
2.5.18 driving the steam valve and the wash water valve of seeding tank and fermentor tank culture transferring pipeline sterilized 15 minutes.
2.5.19 close the vapor cock of culture transferring pipeline on seeding tank and the fermentor tank.
2.5.20 close culture transferring pipeline valve steam valve,
2.5.21 handle by preparing jar working specification after the seeding tank transferred species.
2.6 fermentor cultivation condition and management
2.6.1 temperature: 40 ℃ are not less than 39.5 ℃, patrol and examine once in per 15 minutes, note keeping temperature-stable.
2.6.2. it is serious that tank pressure 0.02mpa-0.06mpa bubbled as the fermentation later stage, can suitably improve tank pressure to 0.06mpa.
2.6.3 air flow quantity: 0-8 hour, 1: 0.8～1: 1.0v/v/min; After 8 hours to putting jar 1: 1.2v/v/min.
2.6.4 the culture transferring amount by volume: 8%
Can not open stirring in 2.6.5 stirring velocity: 0-6 hour; 200rpm after 6 hours.
2.6.6, notice that foam increases according to the fermentation situation, add bubble enemy froth breaking in good time, notice that the top jar is not escaped liquid.
2.6.7 record: per hour once jar is warm for record, air, flow and tank pressure.
2.6.8 sampling: 24 hours mid-early stages of fermenting process every sampling in 8 hours once, carry out sterility test and serial chemical analysis, survey pH, total reducing sugar, amino nitrogen, cell concentration, filtration velocity, microscopy mycelial growth situation has or not microbiological contamination, begin to survey fermentation unit after 24 hours, sampling every two hours once after 24 hours.
2.6.9 fermentation time 40 hours.
2.7 put a jar index
2.7.1 fermentation unit A component reaches 21500 μ g/ml.
2.7.2 fermented liquid residual sugar 2.0%; Reducing sugar 1.2%.
2.7.3 fermented liquid filtering velocity 14ml/min.
2.7.4 the fracture of bacterium mirror mycelia, cavity are in flakes big, pH begins to rise to 7.0.
Embodiment 2 jingganmycin circulating fermentation substratum
Prescription 1: rice meal 9%, jingganmycin wet bacteria slag (moisture content 75%) 11%, soybean cake powder 0.15%, yeast powder 0.7%; Lime carbonate 3%, pH7.2 (before the sterilization).
Prescription 2: rice meal 10%, jingganmycin wet bacteria slag (moisture content 76%) 12%, soybean cake powder 0.15%, yeast powder (0.7%, lime carbonate 4%, pH7.2 (before the sterilization).
Prescription 3: rice meal 9.5%, jingganmycin wet bacteria slag (moisture content 75%) 13%, soybean cake powder 0.15%, yeast powder 0.7%, lime carbonate 3%, pH7.2 (before the sterilization).
Prescription 4: rice meal 10%, jingganmycin wet bacteria slag (moisture content 76%) 14%, soybean cake powder 0.15%, yeast powder 0.7%, lime carbonate 3%, pH7.2 (before the sterilization).
Prescription 5: W-Gum 5%, jingganmycin wet bacteria slag (moisture content 50%) 30%, soybean cake powder 0.1%, groundnut meal 5%, yeast powder 0.5%, lime carbonate 0.5%, pH6.5 (before the sterilization).
Prescription 6: potato starch 20%, jingganmycin wet bacteria slag (moisture content 85%) 1%, soybean cake powder 5%, groundnut meal 0.1%, yeast powder 5%, lime carbonate 5%, pH8 (before the sterilization).
Prescription 7: potato starch 15%, jingganmycin wet bacteria slag (moisture content 85%) 20%, soybean cake powder 0.2%, groundnut meal 0.2%, yeast powder 0.6%, lime carbonate 5%, pH8 (before the sterilization).
3 60 liters of ferment tanks of embodiment
1.1 seed culture medium (each percentage composition in the substratum is weight percentage, down together) rice meal 5.0% peanut meal powder 0.5% yeast powder 0.5%KH
2PO
40.025% CaCO
30.3% NaCl 0.2%PH value nature
1.2 original fermention medium (CK)
Rice meal 10% dregs of beans part 2.5% yeast powder 0.5%
KH
2PO
4 0.30％ CaCO
3 0.1％ NaCl 0.15％ pH7.0
1.3 adopt embodiment 2 prescriptions 1 as jingganmycin wet bacteria slag fermention medium
1.4 technology controlling and process
1.4.1 40 ℃ of seeding tank inoculation secondary fermentation temperature stir revolution 200rpm.
1.4.2 41 hours ferment tank cycles, inoculum size 5%, 40 ℃ of leavening temperatures, tank pressure 0.05Mpa, ventilation: 0-8 hour 1: 1.2v/v/min; 8～41 hours 1: 1.5v/v/min, mixing speed 250rpm.
1.4.3 every filtering bacterium slag of fermentation of taking turns is taken turns one of raw material of fermentation for back one, cleaning of evaporator water that each is taken turns and filter wash water distribution add back one takes turns in the fermentation water.24 hours mid-early stages of fermenting process every sampling in 8 hours once, carry out sterility test and chemical analysis, survey pH, total reducing sugar, amino nitrogen, mycelium concentration, filtration velocity, microscopy mycelial growth situation, have or not microbiological contamination, begin to survey fermentation unit after 24 hours, sampling every two hours once after 24 hours.
1.4.4 put a jar residual sugar to be controlled at＜2.0%, reducing sugar＜1.2%.
Putting jar between the round 1.5 respectively ferment tires and physical and chemical index such as following table 1:
Different round fermentation titers of table 1:NFY-1022 bacterial strain and physical and chemical index
| Test item | ck | 1 takes turns | 2 take turns | 3 take turns | 4 take turns | 4 take turns | 6 take turns | 7 take turns | 8 take turns |
| Initial total reducing sugar g/100ml | 9.8 | 9.5 | 9.0 | 9.3 | 9.4 | 9.6 | 9.4 | 9.2 | 9.0 |
| Put a jar residual sugar g/100ml | 1.60 | 1.65 | 1.70 | 1.73 | 1.80 | 1.56 | 1.90 | 1.95 | 1.98 |
| Put a jar pH | 7.58 | 7.3 | 7.06 | 7.5 | 7.4 | 7.6 | 7.8 | 7.7 | 7.6 |
| Put a jar ammonia nitrogen mg/100ml | 48.74 | 50.0 | 32.0 | 45.0 | 45.0 | 49.0 | 50.0 | 37.0 | 54.0 |
| Filtration velocity ml/5min | 10.5 | 6.0 | 7.9 | 11.0 | 12.0 | 14.0 | 15.0 | 16.0 | 17.0 |
| Put tank volume L | 48.50 | 49.0 | 48.5 | 50.0 | 49.0 | 47.0 | 48.5 | 50.1 | 49.5 |
| Put a jar bacterium slag | 1.45 | 1.55 | 1.65 | 1.70 | 1.80 | 1.90 | 1.95 | 2.00 | 2.00 |
| kg | |||||||||
| This bacterium slag consumption kg | 0.0 | 1.45 | 1.55 | 1.65 | 1.70 | 1.80 | 1.90 | 1.95 | 2.00 |
| Ug/ml tires | 22150 | 25400 | 24580 | 25650 | 24346 | 25000 | 24300 | 24100 | 23150 |
Annotate: tiring is the A component concentration; Put jar bacterium quantity of slag for filtering the back bacterium slag amount of giving money as a gift, unit is KG; Put jar filtration velocity for to filter the clear liquid amount at the appointed time with the volume fermented liquid.
1.6 as can be seen from Table 1, although recycle through 8 rounds, fermentation titer is still compared according to high, on average tire and be 24565ug/ml, comparison is according to high by 10.9%, since each take turns all bacterium slag waste water all by the back one take turns the fermentation used, so can accomplish not have slag and effluent discharge cycle fermentative production jingganmycin.
4 10 tons of ferment tanks of embodiment
2.1 seed culture medium
Rice meal 5.5% peanut meal powder 0.5% yeast powder 0.5%KH
2PO
40.025% CaCO
30.3% NaCl, 0.2% pH value nature
2.2 original fermention medium (CK)
Rice meal 11% bean cake powder 2.5% yeast powder 0.5% KH
2PO
40.30% CaCO
30.5% NaCl, 0.15% pH7.0
2.3 adopt embodiment 2 prescriptions 2 as jingganmycin wet bacteria slag fermention medium
2.4 technology controlling and process
2.4.1 40 ℃ of seeding tank inoculation secondary fermentation temperature stir revolution 180rpm/min.
2.4.2 43 hours ferment tank cycles, inoculum size 8%, 41 ℃ of leavening temperatures, tank pressure 0.03Mpa, ventilation 0～8 hour 1: 1.0; 8～43 hours 1: 1.2, mixing speed 160rpm.
2.4.3 every filtering bacterium slag of fermentation of taking turns is taken turns one of raw material of fermentation for back one, cleaning of evaporator water that each is taken turns and filter wash water distribution add back one takes turns in the fermentation water.24 hours mid-early stages of fermenting process every sampling in 8 hours once, carry out sterility test and chemical analysis, survey pH, total reducing sugar, amino nitrogen, cell concentration, filtration velocity, microscopy mycelial growth situation, have or not microbiological contamination, begin to survey fermentation unit after 24 hours, sampling every two hours once after 24 hours.
2.4.4 put a jar residual sugar to be controlled at＜2.0%, reducing sugar＜1.4%.
Putting jar between the round 2.5 respectively ferment tires and physical and chemical index such as following table 2:
Table 2:NFY-156 bacterial strain is at different round fermentation titers of 10 tons of jars and physical and chemical index
| Test item | ck | 1 takes turns | 2 take turns | 3 take turns | 4 take turns | 4 take turns | 6 take turns | 7 take turns | 8 opinions |
| Initial total reducing sugar g/100ml | 9.32 | 9.20 | 9.15 | 9.00 | 9.45 | 9.50 | 9.35 | 9.34 | 9.46 |
| Put a jar residual sugar g/100ml | 1.67 | 1.60 | 1.75 | 1.45 | 1.70 | 1.78 | 1.90 | 1.94 | 1.90 |
| Put a jar pH | 7.45 | 7.38 | 7.65 | 7.68 | 7.32 | 7.78 | 7.80 | 7.75 | 7.65 |
| Put a jar ammonia nitrogen mg/100ml | 58.74 | 50.0 | 39.0 | 55.0 | 56.0 | 49.0 | 55.0 | 45.0 | 53.0 |
| Filtration velocity ml/5min | 12.5 | 7.0 | 9.9 | 10.0 | 14.0 | 14.5 | 16.0 | 16.0 | 18.0 |
| Put tank volume L | 8000 | 8050 | 8160 | 8180 | 8050 | 8100 | 8050 | 8100 | 815O |
| Put a jar bacterium quantity of slag Kg | 154 | 160 | 165 | 155 | 165 | 170 | 170 | 175 | 175 |
| This bacterium slag consumption Kg | 0 | 154 | 160 | 165 | 155 | 165 | 170 | 170 | 175 |
| Fermentation period hr | 40 | 42 | 42 | 41 | 42 | 41 | 42 | 41 | 43 |
| Ug/ml tires | 22560 | 25800 | 24680 | 24467 | 25489 | 25600 | 24860 | 24500 | 23550 |
Annotate: tiring is the A component concentration; Put jar bacterium quantity of slag for filtering the back bacterium slag amount of giving money as a gift, unit is KG; Put jar filtration velocity for to filter the clear liquid amount at the appointed time with the volume fermented liquid.
2.6 as can be seen from Table 2, although through the recycle of 8 rounds, physical and chemical indexs such as its fermentation titer change similar to 60 liters of fermentor tanks.On average tiring is 24868ug/ml, and comparison is according to high by 10.2%; Since each take turns all bacterium slag waste water all by the back one take turns the fermentation used, so can accomplish not have slag and effluent discharge cycle fermentative production jingganmycin.
5 50 tons of ferment tanks of embodiment
3.1 seed culture medium
Rice meal 5.0% peanut meal powder is yeast powder 0.5%KH O.6%
2PO
4
0.03% CaCO
30.3% NaCl, 0.2% pH value nature
3.2 original fermention medium (CK)
Rice meal 11% bean cake powder 2.5% yeast powder 0.6% KH
2PO
4
0.30％ CaCO
3 0.3％ NaCl 0.15％ pH7.0
3.3 adopt embodiment 2 prescriptions 3 as jingganmycin wet bacteria slag fermention medium
3.4 technology controlling and process
3.4.1 40 ℃ of seeding tank inoculation secondary fermentation temperature stir revolution 160rpm.
3.4.2 42 hours ferment tank cycles, inoculum size 10%, 40 ℃ of leavening temperatures, tank pressure 0.02Mpa, ventilation 0～8 hour 1: 0.8; 8～42 hours 1: 1.25, mixing speed 160rpm.
3.4.3 every filtering bacterium slag of fermentation of taking turns is taken turns one of raw material of fermentation for back one, cleaning of evaporator water that each is taken turns and filter wash water distribution add back one takes turns in the fermentation water.24 hours mid-early stages of fermenting process every sampling in 8 hours once, carry out sterility test and chemical analysis, survey pH, total reducing sugar, amino nitrogen, cell concentration, filtration velocity, microscopy mycelial growth situation, have or not microbiological contamination, begin to survey fermentation unit after 24 hours, sampling every two hours once after 24/ hour.
3.4.4 put a jar residual sugar to be controlled at＜2.0%, reducing sugar＜1.4%.
Putting jar between the round 3.5 respectively ferment tires and physical and chemical index such as following table 3:
Table 3:NFY-1025 bacterial strain is at different round fermentation titers of 50 tons of jars and physical and chemical index
| Test item | ck | 1 takes turns | 2 take turns | 3 take turns | 4 take turns | 4 take turns | 6 take turns | 7 take turns | 8 opinions |
| Initial total reducing sugar g/100ml | 9.5 | 9.25 | 9.55 | 9.50 | 9.45 | 9.50 | 9.55 | 9.34 | 9.56 |
| Put a jar residual sugar g/100ml | 1.57 | 1.65 | 1.85 | 1.45 | 1.60 | 1.88 | 1.90 | 1.96 | 1.95 |
| Put a jar pH | 7.55 | 7.48 | 7.45 | 7.58 | 7.42 | 7.68 | 7.80 | 7.75 | 7.75 |
| Put a jar ammonia nitrogen mg/100ml | 58.74 | 50.0 | 39.0 | 55.0 | 56.0 | 49.0 | 55.0 | 45.0 | 53.0 |
| Filtration velocity ml/5min | 11.5 | 8.0 | 10.9 | 10.0 | 15.0 | 15.5 | 17.0 | 16.5 | 17.5 |
| Put tank volume Lml/5min | 39000 | 40000 | 39800 | 40500 | 40600 | 38900 | 41000 | 39800 | 4100O |
| Put a jar bacterium quantity of slag kg | 741 | 750 | 760 | 766 | 780 | 765 | 770 | 775 | 780 |
| This bacterium slag consumption kg | 0 | 741 | 750 | 760 | 766 | 780 | 765 | 770 | 775 |
| Fermentation period hr | 40 | 41 | 41 | 40 | 41 | 40 | 40 | 40 | 41 |
| Ug/ml tires | 22000 | 25000 | 24650 | 24400 | 25000 | 25000 | 25000 | 25500 | 23000 |
Annotate: tiring is the A component concentration; Put jar bacterium quantity of slag for filtering the back bacterium slag amount of giving money as a gift, unit is KG; Put jar filtration velocity for to filter the clear liquid amount at the appointed time with the volume fermented liquid.
2.6 as can be seen from Table 3, although through the recycle of 8 rounds, physical and chemical indexs such as its fermentation titer change with 10 tons of fermentor tanks wanders seemingly.On average tiring is 24693ug/ml, and comparison is according to high by 12.2%; Since each take turns all bacterium slag waste water all by the back one take turns the fermentation used, so can accomplish not have slag and effluent discharge cycle fermentative production jingganmycin.
6 100 tons of ferment tanks of embodiment
4.1 seed culture medium
Rice meal 5.0% peanut meal powder 0.5% yeast powder 0.5%KH
2PO
4
0.025% CaCO
30.3% NaCl, 0.2% pH value nature
4.2 original fermention medium (CK)
Rice meal 11.5% bean cake powder 2.5% yeast powder 0.5% KH
2PO
4
0.30％ CaCO3 0.5％ NaCl 0.15％ pH7.0
4.3 adopt embodiment 2 prescriptions 4 as jingganmycin wet bacteria slag fermention medium
4.4 technology controlling and process
4.4.1 40 ℃ of seeding tank inoculation secondary fermentation temperature stir revolution 160rpm/min.
4.4.2 40 hours ferment tank cycles, inoculum size 10%, leavening temperature 39-40 ℃, tank pressure 0.02Mpa, ventilation 0～8 hour 1: 1.0; 8～40 hours 1: 1.5, mixing speed 160rpm.
4.4.3 every filtering bacterium slag of fermentation of taking turns is taken turns one of raw material of fermentation for back one, cleaning of evaporator water that each is taken turns and filter wash water distribution add back one takes turns in the fermentation water.24 hours mid-early stages of fermenting process every sampling in 8 hours once, carry out sterility test and chemical analysis, survey pH, total reducing sugar, amino nitrogen, cell concentration, filtration velocity, microscopy mycelial growth situation, have or not microbiological contamination, begin to survey fermentation unit after 24 hours, sampling every two hours once after 24 hours.
4.4.4 put a jar residual sugar to be controlled at＜2.0%, reducing sugar＜1.4%.
Putting jar between the round 4.5 respectively ferment tires and physical and chemical index such as following table 4:
Table 4:NFY-1022 bacterium phosphorus is at different round fermentation titers of 100 tons of jars and physical and chemical index
| Test item | ck | 1 takes turns | 2 take turns | 3 take turns | 4 take turns | 4 take turns | 6 take turns | 7 take turns | 8 opinions |
| Initial total reducing sugar g/100ml | 10.5 | 10.25 | 10.55 | 10.50 | 9.85 | 9.90 | 9.75 | 9.84 | 9.86 |
| Put a jar residual sugar g/100ml | 1.70 | 1.75 | 1.85 | 1.65 | 1.60 | 1.80 | 1.90 | 1.80 | 1.85 |
| Put a jar pH | 7.45 | 7.48 | 7.35 | 7.50 | 7.42 | 7.78 | 7.70 | 7.75 | 7.70 |
| Put a jar ammonia nitrogen mg/100ml | 56.74 | 52.0 | 40.0 | 50.0 | 56.0 | 50.0 | 54.0 | 55.0 | 54.0 |
| Filtration velocity ml/5min | 11.5 | 8.0 | 10.9 | 10.0 | 15.0 | 15.5 | 17.0 | 16.5 | 17.5 |
| Put tank volume L | 79000 | 80000 | 79800 | 80500 | 80600 | 78900 | 80000 | 81000 | 79000 |
| Put a jar bacterium quantity of slag Kg | 1520 | 1550 | 1560 | 1600 | 1650 | 1620 | 1550 | 1600 | 1600 |
| This bacterium slag | 0 | 1520 | 1550 | 1560 | 1600 | 1650 | 1620 | 1550 | 1600 |
| Consumption kg | |||||||||
| Fermentation period hr | 41 | 40 | 41 | 40 | 41 | 40 | 40 | 41 | 42 |
| Ug/ml tires | 22600 | 25600 | 25650 | 24500 | 25600 | 25700 | 25800 | 25000 | 24000 |
Annotate: tiring is the A component concentration; Put jar bacterium quantity of slag for filtering the back bacterium slag amount of giving money as a gift, unit is KG; Put jar filtration velocity for to filter the clear liquid amount at the appointed time with the volume fermented liquid.
2.6 as can be seen from Table 4, although through the recycle of 8 rounds, physical and chemical indexs such as its fermentation titer change similar to 10 tons of fermentor tanks.On average tiring is 25231ug/ml, and comparison is according to high by 11.6%; Since each take turns all bacterium slag waste water all by the back one take turns the fermentation used, so can accomplish not have slag and effluent discharge cycle fermentative production jingganmycin.
Claims (5)
1. the production method of a jingganmycin comprises seed culture, circulating fermentation culture process, it is characterized in that, bacterium slag that last round of fermentation produces in the fermentation culture process and waste water use as the culture medium raw material of next round.
2. method as claimed in claim 1, it is characterized in that, the used substratum of fermentation culture comprises following component by weight percentage: the wet bacteria slag 1～30% of starchy material 5～20%, water content 50～85%, soybean cake powder 0.1～5%, groundnut meal 0.1～5%, yeast powder 0.5～5%, lime carbonate 0.5～5%, surplus is a water, and pH 6.5～8.0.
3. method as claimed in claim 2, wherein said starchy material is selected from one or more in Starch rice, W-Gum, the potato starch.
4. the method for claim 1 is characterized in that the condition of fermentation culture is: 39～42 ℃ of temperature, tank pressure 0.01～0.06Mpa, air flow 1: 0.5～1: 1.5v/v/min, stirring velocity 100～300rpm.
5. as each described method of claim 1～4, it is characterized in that used fermentation strain produces bacterium (Strptomyces hygroscopicusvar.Jingganggensis yen) NFY-1022 bacterial strain for the streptomyces hygroscopicus jingganmycin.
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